Microbial biofilms play an important role in the pathogenesis of various human diseases. Biofilm formation on biomedical devices and implants frequently leads to device failure. Controlling the biofilm growth that leads to disease requires an improved understanding of biofilm development, which in turn requires novel methods of characterizing intact biofilms. Common anti-microbial control strategies such as antibiotics are typically limited in their efficacy at inhibiting or removing biofilms. Quorum sensing species and a wide variety of metabolites exist within biofilms and generally fall within the 2000 Da size range, as do antimicrobial treatments and biomaterial degradation products. The proposed work will develop chemical derivatization, laser desorption postionization mass spectrometry (LDPI-MS), depth profiling, and related methods for the detection of small (<2000 Da) molecular analytes within intact microbial biofilms while preserving information on the spatial distribution of those analytes. Hypothesis: Improved methods of imaging MS of small molecules will lead to improved strategies for the inhibition of bacterial biofilm growth that leads to infections on medical devices and in tissue. Novel imaging MS methods will be developed to probe the resistance of bacterial biofilms to antibiotics and other antimicrobial strategies. The studies will be performed on antibiotic spiked multilayer model films and Staphylococcus epidermidis biofilms. S. epidermidis is a gram positive organism often associated with catheter and hospital infections. It is also biosafety level 1 and therefore can be freely transported and studied at the various laboratories participating in this project. Specific Aim 1: Demonstrate antibiotic and peptide depth profiling on model films. Specific Aim 2: Probe antibiotic resistance in S. epidermidis biofilms. Specific Aim 3: Probe RIP inhibition of S. epidermidis biofilm growth. Specific Aim 4: Probe chitosan inhibition of biofilm growth.